[methods and apparatuses for isolating and preparing stem cells]

ABSTRACT

A method of preparing a population of stem cells for autologous implantation to a subject and apparatus used therein are provided. The cells are activated by irradiating the cells with one or more wavelengths of yellow and red and/or green light. In particular, the cells are irradiated with 575-595 nm (5-20 mW), and 630-635 nm or 660-670 nm (10-100 mW) and/or 510-540 nm (10-60 mW) of monochromatic light for 30-60 mins. Preferably the stem cells are adipose-derived stem cells. Therapeutic applications of the activated stem cells are also provided.

The present application provides methods for preparing stem cells foruse in autologous implantation. The application also provides means ofactivating stem cells and apparatuses that can be used in the methods ofthe invention.

Regenerative medicine harnesses the body's regenerative mechanisms in aclinically targeted manner, using them in ways that are not part of thenormal healing mechanism or by artificially amplifying normalmechanisms.

An example of this process is found in bone marrow transplantation wherehematopoietic stem and progenitor cells are harvested from a donor andplaced into a recipient in whom the normal hematopoietic regenerativemechanisms have been ablated or substantially depleted or impaired,thereby replacing or regenerating the blood-forming capacity of therecipient. In recent clinical and pre-clinical studies this approach hasbeen extended to the non-hematopoietic stem cell component of bonemarrow with studies regenerating (or attempting to regenerate) tissuesincluding bone, heart, and liver. Such work has been based on thedetection of the presence of non-hematopoietic stem cells andendothelial precursor cells in bone marrow.

Such studies used bone marrow transplant recipient animals in whichdonor and host cells could be distinguished by genetic markers to showthat some fraction of new blood vessel development in the recipients wasderived from the donor marrow cells. While this work demonstrates thatmarrow contains such cells, it has generally been extended to mean thatmarrow is therefore the only tissue that contains relevant numbers ofsuch cells, to the extent that when an investigator detects endothelialprecursor cells (EPCs) or marrow stem cells (MSCs) in the circulation itis automatically assumed that these cells are necessarilymarrow-derived. Thus, the concept that cell populations from othertissues might represent an alternative, or perhaps superior, source oftherapeutically relevant cell populations is not addressed.

It has been demonstrated that adipose tissue contains a populationmultipotent stem cells and it has previously shown that this tissue canbe used as a source of endothelial cells, though such studies did notexamine and do not speak in any way to endothelial precursor cells.

Stem cells from embryos or embryonic stem cells (ESCs) are known tobecome many, if not all, of the cell and tissue types of the body. Theseearly foetal cells not only contain all the genetic information of theindividual but also contain the nascent capacity to become any of thecells and tissues of the body. Ongoing research suggests that stem cellshave tremendous scientific and clinical potential.

However, ESCs have theoretic limitations to their use. If usedclinically they would necessarily be derived from another individual,i.e. an embryo. When stem cells or tissues derived from them aretransplanted into another person, toxic immune suppressing drugs may beneeded by the cell recipient to prevent rejection. In addition, anotherindividual's cells can carry viruses or other rare but significantdiseases that can be transmitted to the recipient. Also, ESC-like cells(e.g. teratomas) are known to form tumors.

Recently, non-embryonic or adult stem cells have been identified andrepresent an alternative to the clinical use of ESCs. These cells residein many, if not all, tissues, presumably waiting to respond to trauma orother destructive disease processes so that they can heal the injuredtissue. Emerging scientific evidence indicates that each individualcarries a pool of stem cells that may share with ESCs the ability tobecome many if not all types of cells and tissues.

Adult stem cell populations have been shown to be present in skin,muscle, marrow, liver, brain, and adipose tissue. To date the proposedapplication of such cells in tissue engineering involve increasing cellnumber, purity, and maturity, by processes of cell purification and cellculture. These steps are necessary to compensate for the rarity of stemcells in most tissues. For example, mesenchymal stem cell frequency inbone marrow is estimated at between 1 in 100,000 and 1 in 1,000,000nucleated cells. Similarly, extraction of stem cells from skin involvesa complicated series of cell culture steps over several weeks. Use ofskeletal muscle-derived stem cells in clinical trials of heart diseaseemploys a two to three week culture phase, in which cell number isincreased to clinically relevant numbers and cell differentiation intomuscle is promoted.

These expansion and differentiation steps may provide increased cellnumber, purity, and maturity, but they do so at a cost. This cost caninclude one or more of, loss of cell function due to cell aging, loss ofpotentially useful non-stem cell populations, delays in potentialapplication of cells to patients, increased monetary cost, and increasedrisk of contamination of cells with environmental microorganisms duringculture. While human data is now becoming available with marrow-derivedcells that have not been manipulated but rather used as essentiallywhole marrow, the clinical benefit derived has been suboptimal; anoutcome almost certainly related to the limited cell dose and purityavailable from marrow.

From the above it can be seen that there remains the need for methodsfor preparing stem cell populations for regenerative medicine purposes,in which a population of active cells with increased yield, consistencyand/or purity can be prepared rapidly and reliably, in a cost effectivemanner.

DESCRIPTION OF THE INVENTION

While it is to be understood that the aspects of the invention describedherein can be generally applied to any population of stem cells, thissection of the description discusses adipose-derived stem cells (ADSC)as an example of such stem cells,

The present invention is directed to methods for preparingadipose-derived stem cells (ADSC) from adipose tissue, and methods andsystems for activating ADSC derived from adipose tissue that are placeddirectly into a recipient to promote, engender, or support atherapeutic, structural, or cosmetic benefit.

In one embodiment, adipose tissue processing occurs in a system thatmaintains a closed, sterile fluid/tissue pathway. This is achieved byuse of a pre-assembled, closed sterile container and tubing, allowingfor transfer of tissue and fluid elements within a closed pathway. Aseries of processing reagents (e.g., saline, enzymes, etc.) can beinserted into the container in which the operators manually manage theprocess.

Preferably the entire procedure from tissue extraction throughprocessing and placement into the recipient would all be performed inthe same premises, or more so even within the same room of the patientundergoing the procedure.

For one particular aspect of the invention, raw adipose tissue isprocessed to remove lipid-containing adipocytes and connective tissuethereby obtaining a composition of cells suitable for placement withinthe body of a recipient. The cells are then activated with stimulatorsof cell growth and/or differentiation, optionally derived from growthfactors from the patients own platelets, and/or irradiated withmonochromatic photomodulation. The activated cells, with any of theabove mentioned additives or irradiation, are placed into the personfrom whom they were obtained in the context of an autologous singleoperative procedure with the intention of deriving a therapeutic,structural, or cosmetic benefit to the recipient.

A method of treating a patient includes steps of:

a) removing adipose tissue from a patient using liposuction orlipoplasty in which the adipose tissue has a concentration of stemcells;b) processing the adipose tissue to obtain a higher concentration ofstem cells than before processing, for example by the use of soylecithin;c) activating the final composition of stem cells with stimulators ofstem cells growth and differentiation, for example by the use of use ofautologous platelet-derived growth factors with laser light irradiation;d) administering the activated stem cell composition to a patient.

In another embodiment, a method of treating a patient includes:

a) removing adipose tissue from a patient using liposuction orlipoplasty in which the adipose tissue has a concentration of stemcells;b) processing the adipose tissue to obtain a higher concentration ofstem cells than before processing, for example by the use of soylecithin;c) activating the final composition of stem cells with stimulators ofstem cells growth and differentiation, for example by the use of use ofautologous platelet-derived growth factors with laser light irradiation;d) mixing the adipose tissue having the concentrated stem cells withanother portion of adipose tissue;e) administering the adipose tissue with the increased concentration ofstem cells to a patient.

By administering the activated cells to a patient, one can treatnumerous diseases, including, and not limited to, bone-relateddisorders; adipose related disorders or diseases; liver relateddiseases; myocardial infarctions; renal diseases; retinal diseases;wound healing (e.g., from surgery or diabetic ulcers); skeletal muscledisorders; cartilage and joint repair; lung injuries; diabetes;intestinal disorders; and nervous system disorders, diseases, orinjuries.

The activated stem cells may also be administered to a patient forcosmetic purposes, such as by enhancing physical features, includingreducing wrinkles, enhancing organ mass, and the like.

Hence we disclose a lipid dissolving agent (composed of soy lecithin)added at the end of enzymic digestion of adipose tissue for the purposeof extracting a composition of cells that has an increase concentrationof adipose derived stem cells and a reduced total lipid concentration

We also disclose where activation of extracted adipose-derived stemcells is performed with the patients own platelet-derived growth factors

We also disclose where activation of extracted adipose-derived stemcells is performed by irradiating them with a combination of variousfrequencies (preferably green, yellow and red) of monochromatic light(preferably by laser).

We also disclose methods wherein one or more combinations of the abovestatements are used.

FURTHER DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings.

The present invention may be practiced in conjunction with various cellor tissue separation techniques that are conventionally used in the art,and only so much of the commonly practiced process steps are includedherein as are necessary to provide an understanding of the presentinvention.

While the present invention is generally directed to any population ofstem cells, a preferred embodiment of the invention is directed to acell population present in adipose tissue, and systems and methods foradministering the cell population into a human or animal patient. Thecell population of the adipose tissue may be used as a source of cellsfor therapeutic and cosmetic applications. Among other things, the cellsmay be used for regenerative medicine, such as diseases that can betreated with regenerating cells. The cells of the population may beadministered to a patient without other adipocytes or connective tissue,or may be administered mixed together with adipose tissue in aconcentrated amount, as discussed herein.

It has been discovered that adipose tissue is an especially rich sourceof stem cells. This finding may be due, at least in part, to the ease ofremoval of the major non-stem cell component of adipose tissue, theadipocyte. Thus, in both human and animal studies, processedlipoaspirate (PLA) contains stem cells at a frequency of at least 0.1%,and more typically greater than 0.5%. In certain embodiments of theinvention, PLA has been obtained which contains between about 2-12% stemcells. In even further embodiments, the PLA is processed to obtain apopulation of cells where the stem cells constitute between up to 100%of the cells in the population. The amount of stem cells obtained inaccordance with the invention herein disclosed is substantially greaterthan the published frequency of 1 in 100,000 (0.001%) in marrow(Castro-Malaspina, H., W. Ebell, et al. (1984), Prog Clin Biol Res 154:209-36; Muschler, G. F., H. Nitto, et al. (2001), J Orthop Res 19(1):117-25). Furthermore, collection of adipose tissue is associated withlower morbidity than collection of a similar volume of marrow(Nishimori, M., Y. Yamada, et al. (2002), Blood 99(6): 1995-2001). Inaddition, adipose tissue contains endothelial precursor cells, which arecapable of providing therapy to patients (see for example Masuda, H., C.Kalka, and T. Asahara (2000), Hum Cell. 13(4): p. 153-60; Kaushal, S.,et al., (2001), Nat Med 7(9): p. 1035-40; and Kawamoto, A., et al.(2001) Circulation 103(5): p. 634-7).

The inventors have also determined that it is advantageous to activatestem cells, particularly ADSC, prior to their use for therapeutic andcosmetic applications. As will be described in more detail below, thisactivation can be performed by irradiation of the cells with specificwavelengths of light, and optionally using a mixture of natural orsynthetic growth factors.

A first aspect of the invention provides a method of preparing apopulation of stem cells for autologous implantation to a subject,comprising activating the stem cells by irradiating the cells with oneor more wavelengths of yellow and red and/or green light.

The inventors have determined that it is surprisingly advantageous toirradiate stem cells with one or more specific wavelengths of light,especially monochromatic light, where the light is yellow and red and/orgreen wavelengths of light, prior to their use in autologousimplantation to a subject. While not wishing to be bound to anyparticular theory, and as described further below, the inventorsconsider that specific wavelengths of light act as photomodulators which‘activate’ the stem cells such that the cells are committed to developtowards certain cell fates. In this way, stem cells can be ‘primed’before use such that they can best have a therapeutic and/or cosmeticeffect following autologous implantation.

Preferably, one or more lasers can be used as a source of the light.While yellow light can be used in combination with red or green light,it is preferred that all three are used in the method.

By “yellow”, “red” and “green” light, we include those wavelengths oflight associated with those particular colours. However, preferably inthe method of the first aspect of the invention the followingwavelengths of monochromatic light and power rating are used: 575-595 nm(5-20 mW) (yellow; this can also be considered to be an “orange” rangeof wavelengths as well), and 630-635 nm or 660-670 nm (10-100 mW) (red)and/or 510-540 nm (10-60 mW) (green) for 30-60 mins. An embodiment ofthis aspect of the invention is wherein the cells are irradiated with595 nm (20 mW), 635 nm (60 mW) and 535 nm (60 mW), of monochromaticlight for 30-60 mins.

An embodiment of this aspect of the invention is wherein the methodfurther comprises an initial step of processing a sample of tissue fromthe subject to obtain the population stem cells. Preferably the stemcells are concentrated to form the said population.

The method of the first aspect of the invention can generally be appliedto any population of stem cells.

Stem cells are cells found in most, if not all, multi-cellularorganisms. They are characterized by the ability to renew themselvesthrough mitotic cell division and differentiating into a diverse rangeof specialized cell types. The two broad types of mammalian stem cellsare: embryonic stem cells (ESCs) that are isolated from the inner cellmass of blastocysts; and adult stem cells (ASCs) that are found in adulttissues. In a developing embryo, stem cells can differentiate into allof the specialized embryonic tissues. In adult organisms, stem cells andprogenitor cells act as a repair system for the body, replenishingspecialized cells, but also maintain the normal turnover of regenerativeorgans, such as blood, skin, or intestinal tissues.

Stem cells can now be grown and transformed into specialized cells withcharacteristics consistent with cells of various tissues such as musclesor nerves through cell culture. Highly plastic adult stem cells from avariety of sources, including umbilical cord blood and bone marrow, areroutinely used in medical therapies. Embryonic cell lines and autologousembryonic stem cells generated through therapeutic cloning have alsobeen proposed as promising candidates for future therapies.

Hence by ‘stem cells’, the present application includes both embryonicstem cells and adult stem cells. However, since the method of theinvention relates to the preparation of stem cells for autologousimplant, preferably the stem cells are adult stem cells isolated fromthe patient to be supplied with the prepared stem cells.

By “adult stem cells” we include: adipose-derived stem cells; dermalstem cells; hematopoietic stem cells; mammary stem cells; mesenchymalstem cells; endothelial stem cells; neural stem cells; neural crest stemcells; testicular stem cells.

Adult stem cells been identified in many organs and tissues, includingbrain, bone marrow, peripheral blood, blood vessels, skeletal muscle,skin, teeth, heart, gut, liver, ovarian epithelium, and testis. They arethought to reside in a specific area of each tissue (called a “stem cellniche”). In many tissues, current evidence suggests that some types ofstem cells are pericytes, cells that compose the outermost layer ofsmall blood vessels. Stem cells may remain quiescent (non-dividing) forlong periods of time until they are activated by a normal need for morecells to maintain tissues, or by disease or tissue injury.

Methods of isolating and preparing population of stem cells that can beused in the method of the invention will vary according to the stem celltype to be used, and tissue they are to be isolated from. Many examplesof methods for preparing stem cells from particular tissues are knownand the skilled person would be able to use such methods when preparinga population to be used.

For example, with regard to bone marrow (mesenchymal) stem cells thereare many laboratory methods well known in the art that can be useddirectly or readily adapted so as to provide a population of such stemcells for the invention. Similarly, there are many protocols well knownin the art that can be used to isolate peripheral blood stem cells forthe invention.

A preferred embodiment of this aspect of the invention is where the stemcells are adipose-derived stem cells (ADSC), and the optional step ofprocessing a sample of tissue from a patient to be treated to obtain thepopulation stem cells uses adipose tissue.

A further embodiment of the first aspect of the invention is wherein thestem cells are exposed to one or more growth factors. The growth factorsare Epidermal Growth Factor (EGF); Platelet-Derived Growth Factor(PDGF); Fibroblast Growth Factor (FGFs); Transforming Growth Factors-b(TGFs-b); Erythropoietin (EPO); Insulin-like Growth Factor-I (IGF-I);Insulin-like Growth Factor-II; and/or Tumour Necrosis Factor-a (TNF-a).Preferably the stem cells are exposed to all of the listed growthfactors.

A further embodiment of this aspect of the invention is where the growthfactors are provided by platelet-rich plasma prepared from the subject.As described further below, the inventors have determined thatsurprisingly advantageous to expose the preparation of stem cells togrowth factors before their use in autologous implantation. While notwishing to be bound to any particular theory, and as described furtherbelow, the inventors consider that the growth factors ‘activate’ thestem cells such that the cells towards certain cell fates. In this way,the stem cells can be ‘primed’ before use such that they can best have atherapeutic and/or cosmetic effect following autologous implantation.Preferably the growth factors are provided by platelet-rich plasmaprepared from the subject. Hence the stem cells are only exposed toautologous growth factors prior to their use in implantation, thuslessening the likelihood of infection and immune reactions.

As described further below, in one embodiment of the invention themethod includes where the tissue from the subject is processed to obtaina concentrated population of stem cells.

Preferably the step includes exposing said tissue to a lipid dissolvingagent, preferably lecithin. In addition collagenase could also be usedto disaggregate the tissue so as to prepare the stem cells. Moreover,where the stem cells are ADSC, then the tissue to be used is adiposetissue.

One embodiment of the method of the invention involves adipose tissue asa source of ADSC. Preferably the adipose tissue has been isolated fromthe patient to be treated by liposuction and/or lipoplasty.

A preferred embodiment of the invention is where the method comprisesthe following procedure:

-   -   i) processing a sample of adipose tissue from the subject to        obtain a concentrated population of ADSC, said processing        comprising: removing free lipid and single cell components of        the tissue by rinsing; disaggregating the tissue using        mechanical forces, collagenase and lecithin digestion;        separating and concentrating the ADSC using centrifugation;    -   ii) activating the population of ADSC by irradiating the cells        with 575-595 nm (5-20 mW), and 630-635 nm or 660-670 nm (10-100        mW) and/or 510-540 nm (10-60 mW) of monochromatic light for        30-60 mins, and incubating the cells in the presence of        platelet-rich plasma prepared from the patient to be treated at        30° C. to 38° C. for 5-120 mins.

Preferably the cells are irradiated with 595 nm (20 mW), 635 nm (60 mW)and 535 nm (60 mW), of monochromatic light for 30-60 mins.

An important advantage of this embodiment of the invention is thatsufficient stem cells are prepared from tissue of the subject such thatthe method does not involve a step of culturing the population of stemcells to increase cell number prior to their use in autologousimplantation.

For the avoidance of doubt, the method of the first aspect of theinvention relates to the activation of stem cells for autologousimplantation to a subject, and may not include a step of administeringthe activated stem cells to the subject.

By “subject” we preferably mean a human patient to be treated withautologous implantation to alleviate or treat a particular disorder.Examples of disorders which can be treated with the activated stem cellsof the invention are provided below.

Also for the avoidance of doubt, in each embodiment of the method of theinvention discussed above, it is preferred that the stem cells are ADSC,and where appropriate the tissue is adipose tissue.

A further aspect of the invention provides a population of stem cellsfor autologous implantation obtained by the method of the first aspectof the invention Preferably the stem cells are ADSC.

As discussed above, the stem cells prepared according to the method ofthe invention can be used in methods of treatment. Hence an aspect ofthe invention provides a method of treating a patient using stem cellsfor autologous implantation comprising:

-   -   (i) isolating tissue from the patient;    -   (ii) preparing a population of stem cells according to a method        of the first aspect of the invention; and    -   (iii) administering the activated population of stem cells to        the patient.

Preferably the activated population of stem cells is mixed with afurther portion of tissue prior to administration to the patient.

A preferred embodiment of this aspect of the invention is wherein thetissue is adipose tissue, and the stem cells are ADSC.

A preferred embodiment of this aspect of the invention is wherein saidpatient is treated for therapeutic and/or cosmetic purposes.

A further aspect of the invention provides a population of stem cellsprepared according to the method of the first aspect of the inventionfor use for therapeutic and/or cosmetic purposes. Preferably the stemcells are ADSC.

By “treatment” we include therapeutic and/or cosmetic purposes.Therapeutic purposes includes where the treatment is for bone-relateddisorders; adipose related disorders or diseases; liver relateddiseases; myocardial infarctions; renal diseases; retinal diseases;wound healing; skeletal muscle disorders; cartilage and joint repair;lung injuries; diabetes; intestinal disorders; nervous system disorders,diseases, or injuries; diabetes; alopecia. Preferably the diabetes istype II diabetes.

A still further aspect of the invention provides a method of treating asubject comprising activating stem cells in situ in the subject byirradiating the cells with one or more wavelengths of yellow and redand/or green light. Preferably the following wavelengths ofmonochromatic light and power rating are used: 575-595 nm (5-20 mW)(yellow; this can also be considered to be an “orange” range ofwavelengths as well), and 630-635 nm or 660-670 nm (10-100 mW) (red)and/or 510-540 nm (10-60 mW) (green) for 30-60 mins. An embodiment ofthis aspect of the invention is wherein the cells are irradiated with595 nm (20 mW), 635 nm (60 mW) and 535 nm (60 mW), of monochromaticlight for 30-60 mins.

Examples of disorders which can be treated with this further aspect ofthe invention include the treatment of human diabetic ulcers, in whichthe combination of particular wavelengths of light are applied to theaffected area. While not wishing to be bound to any particular theory,it is thought that the light is activating dermal stem cells in situ inthe subject. Further applications of this aspect of the inventioninclude the irradiation of veins in a subject to be treated, as a meansof affecting immunomodulatory response. Again while not wishing to bebound to any particular theory, it is thought that the light isactivating circulating peripheral stem cells in situ in the subject.

As used herein, “adipose tissue” refers to a tissue containing multiplecell types including adipocytes and microvascular cells. Adipose tissueincludes stem cells and endothelial precursor cells. Accordingly,adipose tissue refers to fat including the connective tissue that storesthe fat.

As used herein, “processed lipoaspirate” (PLA) refers to tissue,preferably adipose tissue, that has been processed to separate theactive cellular component (e.g., the component containing stem cells)from the mature adipocytes and connective tissue. Typically, PLA refersto the pellet of cells obtained by washing and separating the cells fromthe adipose tissue. The pellet is typically obtained by centrifuging asuspension of cells so that the cells aggregate at the bottom of acentrifuge container.

The aspects of the invention may relate to “activated” stem cells,including ADSC. Stem cells can have two states: “quiescent” in which thecells do not reproduce or double and hence do not provide anydifferentiated cell lineages. A second state is “activated”, whichrefers to any stem cell triggered to enter a state of reproduction ordoubling, and can include a cell entering the cell cycle, cell division,or mitosis and providing cell lineages that differentiate into terminalcell types.

There is now provided a detailed description of how the aspects of theinvention can be performed.

Collection of Adipose Tissue

In particular embodiments of the invention disclosed herein, theactivated population of stem cells for autologous implantation to asubject are ADSC and are obtained from adipose tissue. Adipose tissuecan be obtained by any method known to a person of ordinary skill in theart. For example, adipose tissue may be removed from a patient bysuction-assisted lipoplasty, ultrasound-assisted lipoplasty, andexcisional lipectomy. In addition, the procedures may include acombination of such procedures, such as a combination of excisionallipectomy and suction-assisted lipoplasty.

As the tissue or some fraction thereof is intended for autologousimplantation into a patient the adipose tissue should be collected in amanner that preserves the viability of the cellular component and thatminimizes the likelihood of contamination of the tissue with potentiallyinfectious organisms, such as bacteria and/or viruses. Thus, the tissueextraction should be performed in a sterile or aseptic manner tominimize contamination. Suction assisted lipoplasty may be desirable toremove the adipose tissue from a patient as it provides a minimallyinvasive method of collecting tissue with minimal potential for stemcell damage that may be associated with other techniques, such asultrasound assisted lipoplasty.

For suction-assisted lipoplastic procedures, adipose tissue is collectedby insertion of a cannula into or near an adipose tissue depot presentin the patient followed by aspiration of the adipose into a suctiondevice. In one embodiment, a small cannula may be coupled to a syringe,and the adipose tissue may be aspirated using manual force. Using asyringe or other similar device may be desirable to harvest relativelymoderate amounts of adipose tissue (e.g., from 0.1 ml to several hundredmillilitres of adipose tissue). Procedures employing these relativelysmall devices have the advantage that the procedures can be performedwith only local anaesthesia, as opposed to general anaesthesia Largervolumes of adipose tissue above this range (e.g., greater than severalhundred millilitres) may require general anaesthesia at the discretionof the donor and the person performing the collection procedure. Whenlarger volumes of adipose tissue are desired to be removed, relativelylarger cannulas and automated suction devices may be employed in theprocedure.

Excisional lipectomy procedures include, and are not limited to,procedures in which adipose tissue-containing tissues (e.g., skin) isremoved as an incidental part of the procedure, that is, where theprimary purpose of the surgery is the removal of tissue (e.g., skin inbariatric or cosmetic surgery) and in which adipose tissue is removedalong with the tissue of primary interest.

The adipose tissue that is removed from a patient is collected into anapparatus for further processing. As discussed herein, and in oneembodiment, the apparatus is designed for and dedicated to the purposeof collecting tissue for manufacture of a processed adipose tissue cellpopulation, which includes stem cells and/or endothelial precursorcells. In other embodiments, the apparatus may be any conventionalapparatus that is typically used for tissue collection by physiciansperforming the extraction procedure.

The amount of tissue collected will be dependent on a number ofvariables including, but not limited to, the body mass index of thedonor, the availability of accessible adipose tissue harvest sites,concomitant and pre-existing medications and conditions (such asanticoagulant therapy), and the clinical purpose for which the tissue isbeing collected. Experience with transplant of hematopoietic stem cells(bone marrow or umbilical cord blood-derived stem cells used toregenerate the recipient's blood cell-forming capacity) shows thatengraftment is cell dose-dependent with threshold effects. Thus, it islikely that the general principle that “more is better” will be appliedwithin the limits set by other variables and that where feasible theharvest will collect as much tissue as possible.

It has been discovered that the stem cell percentage of 100 ml ofadipose tissue extracted from a lean individual is greater than thatextracted from an obese donor. This reflects a dilutive effect of theincreased fat content in the obese individual. Therefore, it may bedesirable, in accordance with one aspect of the invention, to obtainlarger amounts of tissue from overweight donors compared to the amountsthat would be withdrawn from leaner patients. This observation alsoindicates that the utility of this invention is not limited toindividuals with large amounts of adipose tissue.

Extraction of Adipose-Derived Stem Cells

In particular embodiments of the invention disclosed herein, preparationof the active cell population will require depletion of the fat-ladenadipocyte component of adipose tissue. This is typically achieved by aseries of washing and disaggregation steps in which the tissue is firstrinsed to reduce the presence of free lipids (released from rupturedadipocytes) and peripheral blood elements (released from blood vesselssevered during tissue harvest), and then disaggregated to free intactadipocytes and other cell populations from the connective tissue matrix.In certain embodiments, the entire adipocyte component, or non-stem cellcomponent, is separated from the stem cell component of the adiposetissue. In other embodiments, only a portion or portions of theadipocyte component is separated from the stem cells. Thus, in certainembodiments, the stem cells can be administered with endothelialprecursor cells.

Rinsing is an optional step in which the tissue is mixed with solutionsto wash off free lipid and single cell components, such as thosecomponents in blood, leaving behind intact adipose tissue fragments. Inone embodiment, the adipose tissue that is removed from the patient ismixed with isotonic saline. Intact adipose tissue fragments can beseparated from the free lipid and cells by any means known to persons orordinary skill in the art including, filtration, decantation,sedimentation, or centrifugation.

The intact tissue fragments are then disaggregated using anyconventional techniques or methods, including mechanical force (mincingor shear forces), enzymatic digestion with single or combinatorialprotelolytic enzymes, such as collagenase, trypsin, lipase, liberase H1,as disclosed in U.S. Pat. No. 5,952,215, and pepsin, or a combination ofmechanical and enzymatic methods. For example, the cellular component ofthe intact tissue fragments may be disaggregated by methods usingcollagenase-mediated dissociation of adipose tissue, similar to themethods for collecting microvascular endothelial cells in adiposetissue, as disclosed in U.S. Pat. No. 5,372,945. Additional methodsusing collagenase that may be used in practicing the invention aredisclosed in U.S. Pat. Nos. 5,830,714 and 5,952,215, and by Williams, S.K., S. McKenney, et al. (1995), Cell Transplant 4(3): 281-9. Similarly,a neutral protease may be used instead of collagenase, as disclosed inTwentyman, P. R. and J. M. Yuhas (1980), Cancer Lett 9(3): 225-8.Furthermore, methods of the invention may employ a combination ofenzymes, such as a combination of collagenase and trypsin, as disclosedin Russell, S. W., W. F. Doe, et al. (1976), Int J Cancer 18(3): 322-30;or a combination of an enzyme, such as trypsin, and mechanicaldissociation, as disclosed in Engelholm, S. A., M. Spang-Thomsen, et al.(1985), Br J Cancer 51(1): 93-8.

In a preferred embodiment, the intact tissue fragments are disaggregatedusing mechanical force (mincing and shear forces), enzyme digestion andsoy bean lecithin.

The active cell population (processed lipoaspirate) may then be obtainedfrom the disaggregated tissue fragments by reducing the presence oflipid-containing adipocytes.

Embodiments may employ the use of gravity or a vacuum while maintaininga closed system Separation of the cells in the suspension may beachieved by buoyant density sedimentation, centrifugation, elutriation,differential adherence to and elution from solid phase moieties,antibody-mediated selection, differences in electrical charge;immunomagnetic beads, fluorescence activated cell sorting (FACS), orother means. Examples of these various techniques and devices forperforming the techniques may be found in Hemstreet, G. P., 3rd, P. G.Enoch, et al. (1980), Cancer Res 40(4): 1043-9; Schweitzer, C. M., van,et al. (1995), Exp Hematol 23(1): 41-8; Gryn, J., R. K. Shadduck, et al.(2002), J Hematother Stem Cell Res 11(4): 719-30; Prince, H. M., J.Bashford, et al. (2002), Cytotherapy 4(2): 137-45; Watts, M. J., T. C.Somervaille, et al. (2002), Br J Haematol 118(1): 117-23; Mainwaring, G.and A. F. Rowley (1985), Cell Tissue Res 241(2): 283-90; Greenberg, A.W. and D. A. Hammer (2001), Biotechnol Bioeng 73(2): 111-24; and U.S.Pat. Nos. 6,277,060; 6,221,315; 6,043,066; 6,451,207; 5,641,622; and6,251,295. In the an embodiment, the cells in the suspension areseparated from the cellular component of the suspension using acentrifuge. In one such exemplary embodiment, the cell collectioncontainer may be a flexible bag that is structured to be placed in acentrifuge (e.g., manually or by robotics). In other embodiments, aflexible bag is not used.

After centrifugation, the cellular component forms a pellet, which maythen be resuspended with a buffered solution with or without stimulatorygrowth factors.

In one particular embodiment, the tissue is incubated with collagenaseat a collagenase concentration, temperature, and time sufficient toprovide adequate disaggregation, and then a soy lecithin composition isused, at a preferred concentration, temperature and time sufficient forfurther removal of lipid-containing cells. In a preferred embodiment,the collagenase enzyme used will be approved for human use by therelevant authority (e.g., the U.S. Food and Drug Administration).Suitable collagenase preparations include recombinant andnon-recombinant collagenase. Non-recombinant collagenase may be obtainedfrom F. Hoffmann-La Roche Ltd, Indianapolis, Ind. and/or AdvanceBiofactures Corp., Lynbrook, N.Y. Recombinant collagenase may also beobtained as disclosed in U.S. Pat. No. 6,475,764.

In one embodiment, solutions contain lecithin are incubated at fromabout 30° C. to about 38° C. for from about 5 minutes to about 30minutes. These parameters will vary according to the source of thelecithin, in order to validate that the system is effective atextracting the desired cell populations in an appropriate time frame.

A particular preferred concentration, time and temperature is 20 μg/mlcollagenase (Blendzyme 1, Roche) incubated for 45 minutes, at about 37°C. the lecithin composition incubated for 15 min at about 37° C. Thecollagenase and lecithin used should be free of micro-organisms andcontaminants, such as endotoxin.

Following disaggregation the active cell population may be washed/rinsedto remove additives and/or by-products of the disaggregation process(e.g., collagenase, lecithin and newly-released free lipid). The activecell population could then be concentrated by centrifugation or othermethods known to persons of ordinary skill in the art, as discussedabove. These post-processing wash/concentration steps may be appliedseparately or simultaneously.

In addition to the foregoing, there are many post-wash methods that maybe applied for further purifying the active cell population. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof.

In one embodiment, a solid phase material with adhesive propertiesselected to allow for differential adherence and/or elution of asubpopulation of cells within the processed lipoaspirate is insertedinto the system after the cell washing step. This general approach hasbeen performed in clinical blood transfusion in which filtersdifferentially capturing leukocytes are used to deplete transfused redcells of contaminating white blood cell (Soli, M., et al., 2001, VoxSang 81(2): p. 108-12; Smith, J. W., Apheresis, 1997, Ther Apher. 1(3):p. 203-6). Filters of this type are distributed by Pall Bedical(Leukogard RS and Purecell RCQ) and Asahi (RS2000). Differentialadherence has also been applied to positive selection of monocytes(Berdel, W. E., et al., 1982, Immunobiology 163(5): p. 511-20) andepidermal stem cells (Bickenbach, J. R. and E. Chism, 1998, Exp CellRes. 244(1): p. 184-95).

An alternate embodiment of this differential adherence approach wouldinclude use of antibodies and/or combinations of antibodies recognizingsurface molecules differentially expressed on target and unwanted cells.Selection on the basis of expression of specific cell surface markers(or combinations thereof) is another commonly applied technique in whichantibodies are attached (directly or indirectly) to a solid phasesupport structure (Geiselhart, A., et al., 1996, Nat Immun. 15(5): p.227-33; Formanek, M., et al., 1998, Eur Arch Otorhinolaryngol. 255(4):p. 211-5; Graepler, F., U. Lauer, and M. Gregor, 1998, J Biochem BiophysMethods. 36(2-3): p. 143-55; Kobari et al., 2001, J Hematother Stem CellRes., 10(2): p. 273-81; Mohr, M., et al., 2001, Clin Cancer Res. 7(1):p. 51-7; Pugh, R. E., et al., 1998, J Hematother, 1998. 7(2): p.159-68). This approach has obvious applications in both positive andnegative selection in which, for example, residual white blood cellsmight be removed by use of the CD45 antibody). Similarly, Reyes et alhave applied a complex blend of antibodies in the selection of amultipotential adult progenitor cell from human bone marrow (Reyes, M.,et al. 2001, Blood. 98(9): p. 2615-25). For example, an antibody such asAP2 (Joyner, C. J., et al., 1999, Pathol Res Pract. 195(7): p. 461-6)which specifically binds to adipocytic cells could be employed topreferentially deplete residual adipocytic cells (including immatureadipocytes and adipoblasts). Positive selection could be applied by useof antibodies specific for the target cell population(s). For example,Quirici et al have used antibodies to the Nerve Growth Factor Receptorto enrich bone marrow-derived mesenchymal stem cells (Quirici, N., etal., 2002, Exp Hematol. 30(7): p. 783-91).

In one embodiment of an antibody-based approach, an antibody (forexample AP2) or a cocktail of antibodies (for example AP2, CD3, CD19,CD11b) would be added to the processed lipoaspirate. Many otherantibodies and combinations of antibodies will be recognized by oneskilled in the art and these examples are provided by way of exampleonly. After incubation, under conditions pre-determined to allow foroptimal binding of these antibodies to their cognate antigens, the cellswould be washed by passing through the spinning membrane filter or otherembodiment of the cell washing chamber to remove unbound, excessantibody. The cells would then be passed over a solid phase structuresimilar to that described in the embodiment above but in which the solidphase has attached a secondary antibody capable of high affinityattachment to the primary antibodies now bound to the cell surface.Target cells, for example the adipose tissue-derived stem cell, wouldpass freely through this filter by virtue of the absence of expressionof cell surface antigens recognized by the selected antibody (antibodycocktail) thereby creating a negative selection system.

An antibody-mediated positive selection embodiment could be achieved invery similar fashion by including a third additive that facilitatesdetachment of the cells from the solid phase support. In thisembodiment, the enzyme papain or cymopapain could be added to cleave theantibody molecules and release cells from the solid phase support(Civin, C. I., et al., 1990, Prog Clin Biol Res. 333(387): p. 387-401;discussion 402). Another alternative would be the use of specificpeptides that would compete with the cell surface antigen for binding tothe antibodies, as described by Tseng-Law et al, U.S. Pat. No.6,017,719.

In another embodiment the cell pellet could be resuspended, layered over(or under) a fluid material formed into a continuous or discontinuousdensity gradient and placed in a centrifuge for separation of cellpopulations on the basis of cell density. Examples of media suitable forformation of such gradients include Percoll and Ficoll-Paque (Qian, X.,L. Jin, and R. V. Lloyd, 1998, Endocr Pathol. 9(1): p, 339-346; Smits,G., W. Holzgreve, and S. Hahn, 2000, Arch Gynecol Obstet. 263(4): p.160-3) or Ficoll-Paque (Lehner, M. and W. Holter, 2002, Int Arch AllergyImmunol. 128(1): p. 73-6). Van Merris et al, (Van Merris, V., et al.,2001, Vet Immunol Immunopathol. 83(1-2): p. 11-7) employed adiscontinuous three-step Percoll gradient to separate bovine myeloidcells according to their maturation state on this basis. This embodimentwould be capable of separating out certain residual blood cellpopulations and immature adipocytes (pre-adipocytes) from the cellpopulation.

In a similar embodiment continuous flow approaches such as apheresis(Smith, J. W., Apheresis supra) and elutriation (with or withoutcounter-current) (Lasch, J., G. Kullertz, and J. R. Opalka, 2000, ClinChem Lab Med. 38(7): p. 629-32; Ito, Y. and K. Shinomiya, 2001, J ClinApheresis. 16(4): p. 186-91; Dlubek, D., et al., 2002, Eur J. Haematol.68(5): p. 281-8) may also be employed. Such mechanisms have been used tofractionate blood cells, including separation of red blood cells on thebasis of age (Lasch, J., G. Kullertz, and J. R. Opalka, supra) andapplication of this general approach to further purification of cells ofinterest from processed lipoaspirate will be readily apparent to oneskilled in the art. This embodiment may require modification of theapparatus used to practice the method of the invention such that theapparatus would be integrated with a device providing the apheresis orelutriation capability.

Adherence to plastic followed by a short period of cell expansion hasalso been applied in bone marrow-derived adult stem cell populations(Jaiswal, N., et al., 1997, J Cell Biochem. 64(2): p. 295-312; Hou, L.,et al., 2002, Zhonghua Xue Ye Xue Za Zhi. 23(8): p. 415-9). Thisapproach uses culture conditions to preferentially expand one populationwhile other populations are either maintained (and thereby reduced bydilution with the growing selected cells) or lost due to absence ofrequired growth conditions. Sekiya et al have described conditions whichmight be employed in this regard for bone marrow-derived stem cells(Sekiya, I., et al., 2002, Stem Cells 20(6): p. 530-41). This approach(with or without differential adherence to the tissue culture plastic)could be applied to a further embodiment of this invention. In thisembodiment the cells are removed from the apparatus used for the methodof the invention and placed into a further device providing the cellculture component. This could be in the form of a conventionallaboratory tissue culture incubator or a Bioreactor-style device such asthat described by Tsao et al, U.S. Pat. No. 6,001,642, or by Armstronget al, U.S. Pat. No. 6,238,908.

Activation of the Preparation of Stem Cells

Subsequent to the preparation of a concentrated population of stemcells, the cell population isolated is activated by irradiating thecells with certain frequencies of wavelengths in the visible lightspectrum (400-1200 nm) to stimulated growth and differentiation of stemcells. Light irradiation or photomodulation can be utilized forsignificant benefit in the stimulation of proliferation, growth,differentiation, of stem cells from any living organism. Stem cellsgrowth and differentiation into tissues or organs or structures or cellcultures for infusion, implantation, etc (and their subsequent growthafter such transfer) can be facilitated or enhanced or controlled orinhibited. The origin of such stem cells can be from any living tissueor organism. In humans stem cells for these embodiments may come fromany source in the human body, but typically originate from the bonemarrow, blood, adipose-tissue, embryo, placenta, fetus, umbilical cordor cord blood, and can be either naturally or artificially createdeither in vivo, ex vivo or in vitro with or without genetic alterationor manipulation or engineering. Such tissue can come from any livingsource of any origin.

Stem cells can be photoactivated or photoinhibited by photomodulation.There is little or no temperature rise with this process althoughtransient local nondestructive intracellular thermal changes maycontribute via such effects as membrane changes or structuredconformational changes.

The wavelength or bandwidth of wavelengths is one of the criticalfactors in selective photomodulation. Pulsed or continuous exposure,duration and frequency of pulses (and dark ‘off’ period) and energy arealso factors as well as the presence, absence or deficiency of any orall cofactors, enzymes, catalysts, or other building blocks of theprocess being photomodulated.

Photomodulation can control or direct the path or pathways ofdifferentiation of stem cells, their proliferation and growth, theirmotility and ultimately what they produce or secrete and the specificactivation or inhibition of such production.

Photomodulation can up-regulate or down-regulate a gene or group ofgenes, activate or inactivate enzymes, modulate DNA activity, and othercell regulatory functions.

The selection of wavelength photomodulation is important as is thebandwidth selected as there may be a very narrow bandwidth for someapplications—in essence these are biologically active spectralintervals. Generally the photomodulation will target flavins,cytochromes, iron-sulfur complexes, quinines, heme, enzymes, and othertransition metal ligand bond structures though not limited to these.

These act much like chlorophyll and other pigments in photosynthesis as‘antennae’ for photo acceptor molecules. These photo acceptor sitesreceive photons from electromagnetic sources such as these described inthis application, but also including radio frequency, microwaves,electrical stimulation, magnetic fields, and also may be affected by thestate of polarization of light. Combinations of electromagneticradiation sources may also be used.

The photon energy being received by the photo acceptor molecules fromeven low intensity light therapy (LILT) is sufficient to affect thechemical bonds thus ‘energizing’ the photo acceptor molecules which inturn transfers and may also amplify this energy signal. An ‘electronshuttle’ transports this to ultimately produce ATP (or inhibit) themitochondria thus energizing the cell (for proliferation or secretoryactivities for example). This can be broad or very specific in thecellular response produced. The health of the cells and theirenvironment can greatly affect the response to the photo modulation.Examples include hypoxia, excess or lack or ration of proper cofactorsor growth factors, drug exposure (e.g. reduced ubiquinone from certainanticholesterol drugs) or antioxidant status, diseases, etc. This isanother circumstance wherein oral or systemic replacement of such agentsor factors may be used to enhance the photomodulation effects. It shouldbe also noted that any process which causes the accumulation of suchagents, or conversely accelerates the inactivation or removal ofinhibitors of such agents, would have as a net outcome the effect ofincreasing the concentration of these agents without directly addingsuch agents.

The mechanism, which establishes ‘priorities’ within living cells, canbe photomodulated. This can include the differentiation of early embryosor stem cell population. Exogenous light activated chromophores may alsobe used alone or in combination with exogenous chromophores. Geneticallyaltered or engineered stem cells or stem cells which have an inborngenetic error or defect or uncommon but desirable or beneficial traitmay require a different ‘combination’ of parameters than their analogous‘normal’ stem cells or may produce different cellular response if usethe same combination of parameters. Using various methods ofphotomodulation or other techniques known in the art more specificcellular effects may be produced by ‘blocking’ some ‘channels’ that arephotomodulated.

In the preferred embodiment, the sources of said monochromaticelectromagnetic light could be derived from a light emitting diode, alaser, a fluorescent light source, an organic light emitting diode, alight emitting polymer, a xenon arc lamp, a metal halide lamp, afilamentous light source, an intense pulsed light source, a sulphurlamp, and combinations thereof. The preferred embodiment would be toirradiate the stem cell population with a combination of laser diodesemitting light wavelengths and power ratings: 575-595 nm (5-20 mW)(yellow; this can also be considered to be an “orange” range ofwavelengths as well), and 630-635 nm or 660-670 nm (10-100 mW) (red)and/or 510-540 nm (10-60 mW) (green) for 30-60 mins, where the sample isplaced at a distance of 0-30 cm. More preferably the stem cells areirradiated with 595 nm (20 mW), 635 nm (60 mW) and 535 nm (60 mW), ofmonochromatic light for 30-60 mins. An example of an apparatus which canbe used to irradiate stem cells is provided in the accompanying figures.

The stem cells prepared according to the method of the invention canalso be activated using growth factors that are known to stimulatedgrowth and differentiation of stem cells. Hence an embodiment of theinvention is wherein the stem cells are exposed to one or more growthfactors.

The growth factors can be obtained from a variety of sources: syntheticpeptides; recombinant protein manufacture methods; secretions ofcultured cells; and human or animal tissues. Examples of growth factorswhich can be used in this embodiment of the invention include EpidermalGrowth Factor (EGF); Platelet-Derived Growth Factor (PDGF); FibroblastGrowth Factor (FGFs); Transforming Growth Factors-b (TGFs-b);Transforming Growth Factor-a (TGF-a); Erythropoietin (EPO); Insulin-likeGrowth Factor-I (IGF-I); Insulin-like Growth Factor-II (IGF-II);Interleukin-1 (IL-1); Interleukin-2 (IL-2); Interleukin-3 (IL-3);Interleukin-6 (IL-6); Interleukin-8 (IL-8); Tumour Necrosis Factor-a(TNF-a); Tumour Necrosis Factor-b (TNF-b); Interferon-g (INF-g); ColonyStimulating Factors (CSFs). Such growth factors are well known in theart and can be readily obtained by the skilled person, either fromcommercial suppliers of such peptides, or using commonly appliedtechniques to appropriate biological materials.

Preferably the growth factors are less than 50,000 MW molecules,isolated from porcine fetal mesenchymal stem cell culturemedia/secretions. Analysis of the media by MS/MS showed the followinggrowth factor peptides: Epidermal Growth Factor (EGF); Platelet-DerivedGrowth Factor (PDGF); Fibroblast Growth Factor (FGFs); TransformingGrowth Factors-b (TGFs-b); Erythropoietin (EPO); Insulin-like GrowthFactor-I (IGF-I); Insulin-like Growth Factor-II; Tumour NecrosisFactor-a (TNF-a).

An embodiment of the invention is wherein the growth factors areprovided by platelet-rich plasma prepared from the patient to betreated.

Wound healing is a complex process, involving a mechanism of complexcascading regulatory events at both the molecular and cellular levels.Growth factors (GFs) are secreted by a wide variety of cells to regulatethe wound healing process in an orderly manner. Over the last decade,various GFs, including platelet-derived growth factor (PDGF), andtransforming growth factor-beta (TGF-β), have been used to acceleratethe healing process.

Platelet-rich plasma (PRP), as a storage vehicle of growth factors, is anew application of tissue engineering which was considered for theapplication of growth factors. PRP is a concentration of platelets inplasma developed by gradient density centrifugation. It contains manygrowth factors, such as PDGF, TGF-β, vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF),etc, and it has been successfully used in a variety of clinicalapplications for improving hard and soft tissue healing. Platelet-richplasma has also been shown to enhance the proliferation of humanadipose-derived stem cells.

We provide a protocol below for preparing platelet-rich plasma from thepatient to be treated. Blood would be obtained during or just prior tothe patient's adipose tissue extraction procedure. The method is asfollows:

1. Place blood (20-100 ml) in sterile, anticoagulant containing tubes.2. Mix by inverting all tubes 8-10 times.3. Centrifuge tubes at 72×g for 15 minutes.4. Aspirate top layer (i.e. plasma layer) and transfer aspirate tosterile tubes.5. Centrifuge aspirate at 1000×g for 5 minutes.6 Aspirate plasma to leave a total of 10 ml of plasma with cell pellet.7. Resuspend pellet.8. Irradiate suspension in tubes with Adistem Laser (described below)for 30 min.

9. Centrifuge at 100 g for 5 min.

10. Keep the aspirate.

The stem cells is then mixed with the aspirate and incubated at 30-38°C. (37° C.) for 5-120 min (30 min).

The growth factor components of platelet-rich plasma includes: PDGF(Platelet derived growth factor); TGF-αβ (Transforming growth factoralpha & beta); EGF (Epidermal growth factor) FGF (Fibroblast growthfactor); IGF (Insulin growth factor); PDEGF (platelet derived epidermalgrowth factor); PDAF (platelet derived angiogenesis factor); IL-8(Interleuking-8); TNF-α (Tumor necrosis factor alpha); CTGR (Connectivetissue growth factor) GM-CSF (Granulocyte macrophage colony stimulatingfactor); KGF (Keratinocyte growth factor). The platelet-rich plasma alsoincludes high concentration of leukocytes (neutrophils, eosinophils) formicrobicidal events; high concentration of wound macrophages and otherphagocytic cells, for biological debridement; istamines, Serotonin, ADP,Thromboxane A2, and other vasoactive and chemotactic agents; highplatelet concentration and native fibrinogen concentration for improvedhemostasis.

In another embodiment, the stem cell population isolated is activatedwith both growth factors and light irradiation to stimulate growth anddifferentiation of the stem cells.

The preferred embodiment would be to use the patient's own plateletderived growth factors and combination of light frequencies as describedabove.

Administration of Cells

In certain embodiments, the activated stem cell population isadministered directly into the patient. In other words, the activatedcell population are administered to the patient.

The activated cells that have been concentrated and activated, asdescribed above, may be administered to a patient without furtherprocessing, or may be administered to a patient after being mixed withother tissues or cells. In certain embodiments, where the stem cells areADSC the concentrated activated cells are mixed with adipose tissue thathas not been similarly processed. Thus, by practicing embodiments of themethods of the invention, a composition comprising adipose tissue withan enhanced concentration of active cells may be administered to thepatient. The volumes of the various adipose tissue may be different.

In other embodiments, at least a portion of the activated cellpopulation is stored for later implantation/infusion. The population maybe divided into more than one aliquot or unit such that part of thepopulation of stem cells is retained for later application while part isapplied immediately to the patient. Moderate to long-term storage of allor part of the cells in a cell bank is also within the scope of thisinvention, as disclosed in U.S. patent application Ser. No. 10/242,094,entitled PRESERVATION OF NON EMBRYONIC CELLS FROM NON HEMATOPOIETICTISSUES, filed Sep. 12, 2002, which claims the benefit of U.S.Provisional Patent Application 60/322,070 filed Sep. 14, 2001, which iscommonly assigned, and the contents of which are expressly incorporatedherein by reference. In such an embodiment, the activated cells may bemixed with one or more units of fresh or preserved tissue to provide acomposition containing the stem cells at a higher concentration than aunit of tissue prior to processing.

At the end of processing, the concentrated and activated cells may beloaded into a delivery device, such as a syringe, for placement into therecipient by intradermal, subcutaneous, intravenous, intramuscular, orintraperitoneal techniques. In other words, cells may be placed into thepatient by any means known to persons of ordinary skill in the art, forexample, they may be injected into blood vessels for systemic or localdelivery, into tissue (e.g., cardiac muscle, or skeletal muscle), intothe dermis (subcutaneous), into tissue space (e.g., pericardium orperitoneum), or into tissues (e.g., periurethral emplacement), or otherlocation. Preferred embodiments include placement by needle or catheter,or by direct surgical implantation in association with additives such asa preformed matrix.

The activated cell population may be applied alone or in combinationwith other cells, tissue, tissue fragments, demineralized bone, growthfactors such as insulin or drugs such as members of the thiaglitazonefamily, biologically active or inert compounds, resorbable plasticscaffolds, or other additive intended to enhance the delivery, efficacy,tolerability, or function of the population. The activated cellpopulation may also be modified by insertion of DNA or by placement incell culture in such a way as to change, enhance, or supplement thefunction of the cells for derivation of a cosmetic, structural, ortherapeutic purpose. For example, gene transfer techniques for stemcells are known by persons of ordinary skill in the art, as disclosed inMosca, J. D., J. K. Hendricks, et al., 2000, Clin Orthop 379 Suppl:S71-90, and may include viral transfection techniques, and morespecifically, adeno-associated virus gene transfer techniques, asdisclosed in Walther, W. and U. Stein, 2000, Drugs 60(2): 249-71, andAthanasopoulos, T., S. Fabb, et al., 2000, Int J Mol Med 6(4): 363-75.Non-viral based techniques may also be performed as disclosed inMuramatsu, T., A. Nakamura, et al., 1998, Int J Mol Med 1(1): 55-62.

In one aspect, where the stem cells are ADSC the activated ADSC could bemixed with unprocessed fragments of adipose tissue and placed back intothe recipient using a very large gauge needle or liposuction cannula.Transfer of autologous fat without supplementation with processed cellsis a common procedure in plastic and reconstructive surgery. However,results can be unpredictable as the transferred material tends torapidly reabsorb resulting in an unstable graft. Activated Adiposetissue-derived cells of the invention that are, for example,substantially depleted of mature adipocytes may provide an environmentthat supports prolonged survival and function of the graft.

In another aspect, the activated stem cell population could be placedinto the recipient and surrounded by a resorbable plastic sheath such asthat manufactured by MacroPore Biosurgery, Inc. (U.S. Pat. Nos.6,269,716 and 5,919,234). In this setting the sheath would preventprolapse of muscle and other soft tissue into the area of a bonefracture thereby allowing the emplaced processed adipose tissue-derivedcells to promote repair of the fracture. In this aspect, the beneficialeffect might be enhanced by supplementation with additional componentssuch as pro-osteogenic protein growth factors or biological orartificial scaffolds.

In another aspect, the activated cells could be combined with a geneencoding a pro-osteogenic growth factor which would allow cells to actas their own source of growth factor during bone healing or fusion.Addition of the gene could be by any technology known in the artincluding but not limited to adenoviral transduction, “gene guns,”liposome-mediated transduction, and retrovirus or lentevirus-mediatedtransduction.

Particularly when the activated cells and/or tissue containing the cellsare administered to a patient other than the patient from which thecells and/or tissue were obtained, one or more immunosuppressive agentsmay be administered to the patient receiving the cells and/or tissue toreduce, and preferably prevent, rejection of the transplant. Examples ofimmunosuppressive agents suitable with the methods disclosed hereininclude agents that inhibit T-cell/B-cell co stimulation pathways, suchas agents that interfere with the coupling of T-cells and B-cells viathe CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No.20020182211. Other examples include cyclosporin, myophenylate mofetil,rapamicin, and anti-thymocyte globulin.

In certain embodiments of the invention, the activated cells areadministered to a patient with one or more cellular differentiationagents, such as cytokines and growth factors Examples of various celldifferentiation agents are disclosed in Gimble, J. M., C. Morgan, et al.(1995) J Cell Biochem 58(3): 393-402; Lennon, D. P., S. E. Haynesworth,et al. (1995), Exp Cell Res 219(1): 211-22; Majumdar, M. K., M. A.Thiede, et al. (1998), J Cell Physiol 176(1): 57-66; Caplan, A. I. andV. M. Goldberg (1999), Clin Orthop (367 Suppl): S12-6; Ohgushi, H. andA. I. Caplan (1999), J Biomed Mater Res 48(6): 913-27; Pittenger, M. F.,A. M. Mackay, et al. (1999) Science 284(5411): 143-7; Caplan, A. I. andS. P. Bruder (2001), Trends Mol Med 7(6): 259-64; Fukuda, K. (2001),Artif Organs 25(3): 187-93; Worster, A. A., B. D. Brower-Toland, et al.(2001), J Orthop Res 19(4): 738-49; Zuk, P. A., M. Zhu, et al. (2001),Tissue Eng 7(2): 211-28; and Mizuno, H., P. A. Zuk, et al. (2002), PlastReconstr Surg 109(1): 199-209; discussion 210-1.

By administering the activated cells to a patient, one can treatnumerous diseases, including, and not limited to, bone-relateddisorders, diseases, or injuries, including slow/non-union fractures,osteoporosis (age-related or chemotherapy-induced), inherited diseasesof bone (osteogenesis imperfecta); adipose related disorders ordiseases; liver related diseases, disorders, or injuries, includingliver failure, hepatitis B, and hepatitis C; myocardial infarctions,including heart attack or chronic heart failures; renal diseases orkidney damage; retinal diseases or damage or necrosis; wound healing(e.g., from surgery or diabetic ulcers); skeletal muscle disorders bothtraumatic and inherited; cartilage and joint repair both traumatic andautoimmune; lung injuries; diabetes; intestinal disorders; nervoussystem disorders, diseases, or injuries, such as central nervous systemsdisorders, diseases, or injuries, including spinal cord injuries,Parkinson's disease, Alzheimer's disease, and stroke.

The activated stem cells may also be administered to a patient forcosmetic purposes, such as by enhancing or improving physical features,including reducing wrinkles, enhancing organ mass, and the like.

Many other conformations of the staged mechanisms used for cellprocessing will be apparent to one skilled in the art and the presentdescription is included as one example only. For example, mixing oftissue and saline during washing and disaggregation may occur byagitation as in the present example or by fluid recirculation. Cellwashing may be mediated by a continuous flow mechanism such as thespinning membrane approach, differential adherence, differentialcentrifugation (including, but not limited to differentialsedimentation, velocity, or gradient separation), or by a combination ofmeans. Similarly, additional components to allow further manipulation ofcells including addition of growth factors or other biological responsemodifiers (Lind, M., (1998) Acta Orthop Scand Suppl, 1998. 283: p. 2-37;Hanada, K., J. E. Dennis, and A. I. Caplan, (1997) J Bone Miner Res.12(10): p. 1606-14; Lieberman, J. R., et al., (1998) J Orthop Res,16(3): p. 330-9), mixing of cells with other structural components(e.g., bone fragments (Jean, J. L., S. J. Wang, and M. K. Au (1997) JFormos Med Assoc 96(7): p. 553-7), collagen (Saadeh, P. B., et al.,(2001) J Craniofac Surg. 12(6): p. 573-579) and/or synthetic componentsintended for implant with the cells into the recipient (Petite, H., etal., (2000) Nat Biotechnol., 18(9): p. 959-63.taf/dynapage.taf?file=incb/biotech/v18/n9/full/nbt0900.sub.—959.htmltaf/dynapage.taf?file=mcb/biotech/v18/n9/abs/nbt0900.sub.—959.html; Gao,J., et al., (2001) Tissue Eng. 7(4): p. 363-71; Ohgushi, H. and A. I.Caplan, supra; Caplan, A. I. and V. M. Goldberg, supra). Post-processingmanipulation may also include cell culture (Caplan, A. I. and S. P.Bruder, supra; Petite, supra; Zuk, P. A., et al., 2001 supra) genetransfer (Luskey, B. D., et al. (1990) Ann N Y Acad Sci. 612(398): p.398-406; Grompe, M., et al. (1998) J Inherit Metab Dis. 21(5): p.518-31; Gazit, D., et al. (1999) J Gene Med. 1(2): p. 121-33; Mosca, J.D., et al., supra), or further cell purification (Greenberg, A. W. andD. A. Hammer, 2001 supra; Mainwaring, G. and A. F. Rowley, 1985 supra;Schweitzer, C. M., et al., 1995 supra).

In additional embodiments of the invention relating to ADSC, tissuecollected into a conventional adipose tissue trap could be transferredinto a processing set designed for processing other tissues. Forexample, Baxter Inc. manufacture and sell a series of plastic bags andfilters intended for use in the setting of a bone marrow transplantharvest (“Bone Marrow Collection Kit with Flexible Pre-Filters andInline Filters”, Product Code, 4R2107, U.S. Pat. Nos. 4,346,703 and5,724,988). This bag set contains a large conical bag with an integrated800 μm filter which could be used for washing the collected adiposetissue. In this example adipose tissue fragments larger than 800 μmwould be retained in the bag. These fragments could then be washed byrepeated addition of saline (or other washing solution) followed byremoval of waste material through ports below the filter. Mixing couldbe achieved manually or by use of a benchtop rocking device and warmingcould be applied by use of a heating pad. Disaggregation could occurwithin the lumen of this bag. Following disaggregation cells would passthrough the integrated 800 μm filter (and optionally through one or morefilters of smaller mesh size provided with the kit) and collected into acollection bag (also provided). This bag could then be placed into acentrifuge (e.g., a Sorval RC-3C) where cells could be serially washedand concentrated. Cells could also be washed using existing cell washingdevices (largely developed for washing human blood products) such asthose sold by Baxter Inc (Cytomate or Baxter CS3000) or by Cobe Inc.(Cobe Spectra). The disposable elements may be integrated using thefittings provided by the manufacturer or they may be linked by use of asterile connecting device such as those manufactured by Terumo Inc.Similarly the mechanisms described in this less integrated approachcould be linked to a central controller and assembled as components of amore integrated device. A peristaltic pump or battery of pumps could beused to automate fluid flow with use of manual or automated clamping toopen and close fluid pathways.

Alternatively, a plastic bag (similar to an i.v. saline bag) withvarious inlets similar to what they use for placental cord blood can beused in the method of the invention, for example bags available from acompany called Fresenius.

In a preferred embodiment of the invention, the tissue removal systemand processing set would be present in the vicinity of the patientreceiving the treatment, such as the operating room or out-patientprocedure room (effectively at the patient's bedside). This allowsrapid, efficient tissue harvest and processing, alleviating theopportunity for specimen handling/labeling error and thereby allow forperformance of the entire process in the course of a single surgicalprocedure.

Once prepared, the activated stem cells can be labelled prior toadministration to a subject. This allows a physician to determine tolocation of the administered cells in the patient once administered.Preferably the activated stem cells are labelled with 99Tc HMPAO; anexperimental protocol for ADSC labelling is provided in the accompanyingexamples.

A further aspect of the invention provides an apparatus for thepreparation of a population of stem cells for autologous implantation,said apparatus comprising:

-   -   i) a tissue preparation container including a one way inlet port        structured to receive tissue removed from a patient and for the        administration of at least one additive to mix with the stem        cells contained therein;    -   ii) an outlet structured to permit the cells in the container to        be removed.

A preferred embodiment of the apparatus of the invention is wherein theapparatus includes means for mechanically disrupting the tissue.Preferably the apparatus also includes means for separating, andoptionally concentrating, the stem cells from further components of thetissue.

A further preferred embodiment of this aspect of the invention iswherein the apparatus further comprises:

-   -   i) one or more sources of wavelengths of yellow and red and/or        green light; and    -   ii) a locator for arranging a population of cells in the said        wavelengths of light.

A further aspect of the invention provides an apparatus for preparing anactivated population of stem cells for autologous implantation to asubject, comprising:

-   -   i) one or more sources of wavelengths of yellow and red and/or        green light; and,    -   ii) a locator for arranging a population of cells in the said        wavelengths of light.

In the apparatus of the aspects of the invention, it is preferred thatthe wavelengths are 575-595 nm (5-20 mW), and 630-635 nm or 660-670 nm(10-100 mW) and/or 510-540 nm (10-60 mW) of monochromatic light; mostpreferably 595 nm (20 mW), 635 nm (60 mW) and 535 nm (60 mW) ofmonochromatic light.

Also, in the apparatus of the aspects of the invention, it is preferredthat the stem cells are ADSC and where appropriate the tissue is adiposetissue.

The following examples are provided to demonstrate particular situationsand settings in which this technology may be applied and are notintended to restrict the scope of the invention and the claims includedin this disclosure

FIG. 1: A schematic diagram of the procedure for autologous implantationof activated ADSC.

FIG. 2: Details of the ADSC harvested by the procedures disclosedherein.

FIG. 3: Examples of stem cell regulators which can be used to activatethe ADSL.

FIG. 4: Table demonstrating the light activation of ADSC. Five frequencyranges (2 in the green, 1 in the yellow an 2 in the red) where found tostimulate adipose-derived MSCs.

FIG. 5: Effect of intradermal injections of autologous adipose-derivedstromal cells for cosmetic application in alopecia

FIG. 6: Effect of intravenous administration of autologousadipose-derived stem cells on diabetic neuropathic ulcers

FIG. 7: Case Study: Type II Diabetic. Intravenous treatment withactivated autologous adipose-derived stem cells.

FIG. 8: Case Study: Type II Diabetic. Intravenous treatment withactivated autologous adipose-derived stem cells.

FIG. 9: Diagram of an apparatus for irradiating ADSC with one or morewavelengths of light.

EXAMPLE 1 Autologous Fat Transfer

Autologous fat transfer is a relatively common cosmetic and structuralprocedure involving the harvest of adipose tissue (fat) from onelocation and reimplantation in another location within the sameindividual (Coleman, S. R. (1995) Aesthetic Plast Surg 19(5): 421-5;Coleman, S. R. (2001) Clin Plast Surg 28(1): 111-9; Coleman, W. P., 3rd(1991) Plast Reconstr Surg 88(4): 736). However, as indicated above,this procedure is frequently compromised by inconsistent engraftmentsuch that the implanted material is fully or partially resorbed or isreplaced by scar tissue (Eremia, S. and N. Newman (2000), Dermatol Surg26(12): 1150-8). At least part of the loss of function can be attributedto necrosis of implanted fat tissue during the time it takes for newblood vessels to form and feed the implant. Thus tissue implanted intohighly vascular areas such as muscle beds shows better engraftment thanwhen implanted into less well perfused tissues (Guerrerosantos, J., A.Gonzalez-Mendoza, et al. (1996) Aesthetic Plast Surg 20(5): 403-8).

Processed lipoaspirate prepared as described in this disclosureaddresses this issue by supplementing the implant with additionalendothelial precursors and stem cells. In a clinical application of thistechnology, processed lipoaspirate derived according to this disclosureis prepared and mixed with intact (non-disaggregated) adipose tissuefragments, as disclosed above. The composition comprising the mixture ofadipose tissue and the activated stem cells may be implanted into therecipient to provide an autologous soft tissue filler for correction ofcontour defects (wrinkles, “divots,” pockmarks, and larger deficits)(Coleman, S. R. (2001) supra) or for providing support to damagedstructures such as the urethra (Palma, P. C., C. L. Riccetto, et al.(1997) J Endourol 11(1): 67-70; Lee, P. E., R. C. Kung, et al. (2001) JUrol 165(1): 153-8). It can also be use for breast and penileaugmentation, as well as a filler in a breast that has had a cancerlumpectomy.

EXAMPLE 2 Type II Diabetes

A clinical trial on the intravenous administration of activated stemcells prepared as in embodiment above has been performed. The title ofthe trial was: Safety and efficacy of autologous adipose-derived stromalcells on Type II diabetes patients: 6 month post procedure results. Somedata from the trial is shown in the accompanying figures. The abstractof the trial was:

“Stem cell therapies hold great promise for anti-aging benefits as theyare regenerative in nature. Autologous adipose-derived stem celltransplants hold even more potential as they have no ethical barriersand require no out-of-surgery culture requirements. We have devised aprocedure that entails the isolation of stromal cells fromadipose-tissue derived from a mini-liposuction procedure, theiractivation from a quiescent stage to an active stage, and theirreintroduction back into the patient via intravenous mode. This singleprocedure has now been performed on 176 subjects over a two and a halfyear period in four countries with no adverse effect. Because these wereisolated case studies a formal clinical trial was then initiated toassess the safety and efficacy of the procedure on a controlled group of34 patients with non-insulin and insulin-dependent type II diabetesmellitus with no cardiovascular or nephrological complications. Afterthree months post-operation the patients showed a significant andsustained reduction in fasting glucose levels (from 9.64+3.88 mmol/1 to7.01+1.64 mmol/l; p=0.005 at 2 weeks to 7.71+2.29 mmol/l; p=0.01 at 12weeks), glycosylated haemoglobin (from 9.11+2.06% to 7.73+1.19%;p=0.00001), C-peptide (from 2.75+1.02 to 2.27+1.45; p=0.045) andtriglycerides (from 2.31+1.53 to 1.91+1.63; p=0.03). At six months postprocedure nearly half the patients reverted back to pre-op conditionswhile the other half continues to see sustained decreases in diabeticparameters as compared to pre-op levels. Six month statistics showedFasting blood sugar went from 9.64+3.88 mmol/1 at pre-op to 8.50+2.86mmol/l; p=ns at 24 weeks), glycosylated haemoglobin (from 9.11+2.06% to8.10+1.82%; p=0.001), C-peptide (from 2.75+1.02 to 2.83+1.37; p=ns) andtriglycerides (from 2.31+1.53 to 2.01+1.35; p=ns). Most patients havenoticed an increase in well-being parameters post-op. There was nosignificant change detected post-op in total cholesterols and other CBC,LFT and KFT values and no obvious adverse reaction has been noted. Theresults of the trial to date suggest that the autologous adipose derivedstromal cell therapy appears to be safe and beneficial to type IIdiabetes patients by decreasing their resistance to insulin anddecreasing diabetic cardiovascular risk factors. We believe that thestromal cell transplant is probably acting by increasing adiponectinlevels in these subjects, an adipocytokine that is produced by adiposestromal cells and known to regulate insulin-resistance. Lifestyle andhypoglycemic medication changes also plays an important role insustaining the effects observed.” FIG. 3 shows statistical results.

EXAMPLE 3 Alopecia

Subcutaneous and intradermal administration of activated stem cells(prepared as in the described embodiment above) into the scalp of menwith male pattern baldness has been performed. Increased folliclethickness, colouration, and new hair follicle growth has been observedin a number of cases. No adverse reaction has yet to be noticed. Anillustrative example of this is provided in the accompanying figures.

EXAMPLE 4 99Tc HMPAO Labelling of Adipose Derived Stromal VascularFraction Cells

You will need the following:

1) hexamethylpropylene amine oxime (HMPAO) vials from Ceretec.

2) Tc99m (370 MBq)

3) sterile 15% NaCl solution4) sterile Saline5) 2 ml sterile centrifuge (eppendorf) tubes6) 15 ml sterile plastic tubes screw-top.1. Take 10% of the stromal vascular fraction to be injected.2. Spin the sample at 600×g for 5 min. Remove the supernatant.3. For lysis of re blood cells add 10 ml hypertonic saline—NaCl 1.8%(8.8 ml saline+1.2 ml NaCl 15%)

4. Let sit for 30 sec.

5. Spin at 600×g for 5 min and remove supernatant.6. Reconstitute pellet with 0.5 ml Saline.7. Add to the Ceretec tube 1.5 nil saline and vortex 5-6 times.8. Take 0.5 ml of Ceretec and add 370 MBq Tc99m. Vortex 5-6 with over a30 min period.9. Ad this mixture to the reconstituted stromal vascular fraction cells.Let this sit for 15 min, with gentle shaking every 2-3 min.10. Spin the mixture at 600×g for 5 min. Wash with saline an re-spin at600×g for 5 min. Repeat once more. Reconstitute cells in 2 ml saline.11. With a two ml syringe inject the cells by i.v. with a 1-2 minperiod.12. Observe the patient in case of allergic symptoms.13. Do a full body gamma scan on patient 12-24 hrs later.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. For purposes of summarizing thepresent invention, certain aspects, advantages and novel features of thepresent invention have been described herein. Of course, it is to beunderstood that not necessarily all such aspects, advantages or featureswill be embodied in any particular embodiment of the present invention.Additional advantages and aspects of the present invention are apparentin the following detailed description and claims.

The above-described embodiments have been provided by way of example,and the present invention is not limited to these examples. Multiplevariations and modification to the disclosed embodiments will occur, tothe extent not mutually exclusive, to those skilled in the art uponconsideration of the foregoing description. Additionally, othercombinations, omissions, substitutions and modifications will beapparent to the skilled artisan in view of the disclosure herein.Accordingly, the present invention is not intended to be limited by thedisclosed embodiments, but is to be defined by reference to the appendedclaims.

A number of publications and patents have been cited hereinabove. Eachof the cited publications and patents are hereby incorporated byreference in their entireties.

1-35. (canceled)
 36. A method of preparing a population of stem cellsfor autologous implantation to a subject, comprising activating the stemcells by irradiating the cells with one or more wavelengths of yellowand red and/or green light.
 37. The method of claim 36, wherein yellowand red and green light is used.
 38. The method of claim 36, wherein thecells are irradiated with 575-595 nm (5-20 mW), and 630-635 nm (10-100mW) and/or 510-540 nm (10-60 mW) of monochromatic light for 30-60 mins.39. The method of claim 38, wherein the cells are irradiated with 595 nm(20 mW), 635 nm (60 mW) and 535 nm (60 mW) of monochromatic light, for30-60 mins.
 40. The method of claim 36, comprising a further initialstep of processing a sample of tissue from the subject to obtain thepopulation stem cells to be irradiated.
 41. The method of claim 36,wherein the stem cells are adult stem cells.
 42. The method of claim 41,wherein the adult stem cells are adipose-derived stem cells (ADSC);dermal stem cells; hematopoietic stem cells; mammary stem cells;mesenchymal stem cells; endothelial stem cells; neural stem cells;neural crest stem cells; or testicular stem cells.
 43. The method ofclaim 36, wherein the stem cells are ADSC and the tissue is adiposetissue.
 44. The method of claim 36, wherein the stem cells are exposedto one or more growth factors.
 45. The method of claim 44, wherein thegrowth factors are Epidermal Growth Factor (EGF); Platelet-DerivedGrowth Factor (PDGF); Fibroblast Growth Factor (FGFs); TransformingGrowth Factors-b (TGFs-b); Erythropoietin (EPO); Insulin-like GrowthFactor-I (IGF-I); Insulin-like Growth Factor-II; and/or Tumour NecrosisFactor-a (TNF-a).
 46. The method of claim 44, wherein the growth factorsare provided by platelet-rich plasma prepared from the subject.
 47. Themethod of claim 43, wherein the step of processing the sample of adiposetissue includes exposing said tissue to a lipid dissolving agent. 48.The method of claim 47, wherein said lipid dissolving agent is lecithin.49. The method of claim 43, wherein the step of processing the sample ofadipose tissue includes exposing said tissue to collagenase.
 50. Themethod of claim 40, wherein where the tissue is adipose tissue, then theadipose tissue has been isolated from the subject by liposuction and/orlipoplasty.
 51. The method of claim 43, comprising: i) processing asample of adipose tissue from the subject to obtain a concentratedpopulation of ADSC, said processing comprising: removing free lipid andsingle cell components of the tissue by rinsing; disaggregating thetissue using mechanical forces, collagenase and lecithin digestion;separating and concentrating the ADSC using centrifugation; ii)activating the population of ADSC by irradiating the cells with 575-595nm (5-20 mW), and 630-635 nm (10-100 mW) and/or 510-540 nm (10-60 mW) ofmonochromatic light for 30-60 mins, and incubating the cells in thepresence of platelet-rich plasma prepared from the patient to be treatedat 30° C. to 38° C. for 5-120 mins.
 52. The method of claim 36, whereinsaid method does not involve a step of culturing the population of stemcells to increase cell number prior to their use in autologousimplantation.
 53. A population of stem cells for autologous implantationobtained by the method of claim
 36. 54. A method of treating a patientusing stem cells for autologous implantation comprising: (i) isolatingtissue from the patient; (ii) preparing a population of stem cellsaccording to a method of claim 36; and (iii) administering the activatedpopulation of stem cells to the patient.
 55. The method of claim 54,wherein the population of stem cells is mixed with a further portion oftissue prior to administration to the patient.
 56. The method of claim54, wherein the stem cells are ADSC and the tissue is adipose tissue.57. The method of claim 54, wherein said patient is treated fortherapeutic and/or cosmetic purposes.
 58. A population of stem cellsprepared according to the method of claim 36 for therapeutic and/orcosmetic purposes.
 59. A population of stem cells prepared according tothe method of claim 54 for therapeutic and/or cosmetic purposes.
 60. Themethod of claim 54, wherein said treatment is for bone-relateddisorders; adipose related disorders or diseases; liver relateddiseases; myocardial infarctions; renal diseases; retinal diseases;wound healing; skeletal muscle disorders; cartilage and joint repair;lung injuries; diabetes; intestinal disorders; nervous system disorders,diseases, or injuries; diabetes; alopecia.
 61. The method of claim 60,wherein the treatment is for type II diabetes.
 62. A method of treatinga subject comprising activating stem cells in situ in the subject byirradiating the cells with one or more wavelengths of yellow and redand/or green light.
 63. The method of claim 62, wherein the cells areirradiated in situ in the subject with 575-595 nm (5-20 mW), and 630-635nm (10-100 mW) and/or 510-540 nm (10-60 mW) of monochromatic light for30-60 mins.
 64. The method of claim 63, wherein the cells are irradiatedin situ in the subject with 595 nm (20 mW), 635 nm (60 mW) and 535 nm(60 mW) of monochromatic light, for 30-60 mins.
 65. An apparatus for thepreparation of a population of stem cells for autologous implantation,said apparatus comprising: i) a tissue preparation container including aone way inlet port structured to receive tissue removed from a patientand for the administration of at least one additive to mix with the stemcells contained therein; ii) an outlet structured to permit the cells inthe container to be removed.
 66. The apparatus of claim 65, furthercomprising means for mechanically disrupting the tissue.
 67. Theapparatus of claim 65, further comprising means for separating, andoptionally concentrating, the stem cells from further components of thetissue.
 68. The apparatus of claim 65, further comprising i) one or moresources of wavelengths of yellow and red and/or green light; and, ii) alocator for arranging a population of cells in the said wavelengths oflight.
 69. An apparatus for preparing an activated population of stemcells for autologous implantation to a subject, comprising: i) one ormore sources of wavelengths of yellow and red and/or green light; and,ii) a locator for arranging a population of cells in the saidwavelengths of light.
 70. The apparatus of claim 68, wherein thewavelengths are 575-595 nm (5-20 mW), and 630-635 nm (10-100 mW) and/or510-540 nm (10-60 mW) of monochromatic light; most preferably 595 nm (20mW), 635 nm (60 mW) and 535 nm (60 mW) of monochromatic light.
 71. Theapparatus of claim 69, wherein the wavelengths are 575-595 nm (5-20 mW),and 630-635 nm (10-100 mW) and/or 510-540 nm (10-60 mW) of monochromaticlight; most preferably 595 nm (20 mW), 635 nm (60 mW) and 535 nm (60 mW)of monochromatic light.
 72. The apparatus of claim 65, wherein the stemcells are ADSC and the tissue is adipose tissue.
 73. The apparatus ofclaim 69, wherein the stem cells are ADSC and the tissue is adiposetissue.